Maryelle Kolopp

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure).The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs.

Poly(ethylene carbonate)s, part II: degradation mechanisms and parenteral delivery of bioactive agents.

The degradation and drug carrier properties of poly(ethylene carbonate) (PEC) were investigated in vitro and in rats and rabbits. PEC was found to be specifically degraded in vivo and in vitro by superoxide radical anions O2-*, which are, in vivo, mostly produced by inflammatory cells. No degradation of PEC was observed in the presence of hydrolases, serum or blood. PEC is biodegraded by surface erosion without significant change in the molecular weight of the residual polymer mass. The non-hydrolytic biodegradation by cells producing O2-* is unique among the polymers used as biodegradable drug carriers. The main degradation product of PEC in aqueous systems is ethylene glycol, formed presumably by hydrolysis of ethylene carbonate. The splitting off of a five-membered ring structure from the polymer chain indicates a chain reaction mechanism for the biodegradation. PEC is a suitable drug carrier, particularly for labile drugs. Using human interleukin-3 and octreotide as model drugs, surface erosion of the PEC formulations was indicated by a 1:1 correlation between drug release and polymer mass loss.

Assessment of the Phototoxic Potential of Compounds and Finished Topical Products Using a Human Reconstructed Epidermis

The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.

Morphologic and Immunohistochemical Characterization of Leydig Cell Tumor Variants in Wistar Rats

During a routine long-term drug safety study, lasting approximately 2½ yr, male Wistar rats, treated with a prolactin-inhibiting compound, developed an excess of Leydig cell tumors (LCTs). Most tumors were typical for the rat but a small number showed an unusual variation and some appeared malignant. The variation consisted of glandular and/or tubular structures within the tumor mass which occasionally anastomosed and contained an eosinophilic periodic-acid Schiff (PAS) positive material. In a few of these variants, malignant features such as cellular atypia, capsular, and lymphatic invasion and necrosis were seen. No metastases were detected. Detailed morphological and immunohistochemical investigations were conducted in order to establish the cell of origin of these variants. Glandular/tubular structures were found to stain with varying intensity for vimentin and cytokeratin, but were always negative for β-tubulin. The results indicated that the cell of origin of these LCT variants was indeed the Leydig cell and that glandular and/or tubular structures within LCTs represented a form of Leydig cell metaplasia.

In vitro tolerability of human nasal mucosa: histopathological and scanning electron-microscopic evaluation of nasal forms containing Sandostatin®

Anin vitro human nasal model was developed as a tool to study the local tolerabiliity of nasal powder forms using excised nasal mucosa in a diffusion chamber. The suitability of this model was tested using Sandostatin® (SMS) an octapeptide analog of somatostatin, as a reference drug enhanced by Avicel® (microcrystalline cellulose) or lactose (100 mesh). The standard nasal spray vehicle was taken as a harmless control and 1% chenodeoxycholate (CDC) as a harmful control in terms of local tolerability. The extent of peptide permeation was determined by measuring SMS concentration in the receiving chamber. The labeling of SMS was detected by immunoperoxidase staining on cross sections. The local tolerability for all tested forms was assessed by histopathological examination and scanning electron microscopy. The apparent permeation coefficient allowed us to rank the absorption of the tested drug forms as Avicel > spray=lactose>1%CDC. For all formulations, SMS was detected in the epithelium. No changes of the nasal mucosa could be observed with Avicel, lactose or nasal spray vehicle in the presence or absence of SMS. 1%CDC with or without drug showed an immediate destruction of the nasal epithelium. The validation of thisin vitro model using human nasal mucosa will be further discussed as a tool for assessing the local tolerability of intranasally applied test substances.

A brief review of modern toxicologic pathology in regulatory and explanatory toxicity studies of chemicals

Macroscopic and histologic evaluation of animal studies for general toxicity and carcinogenicity are cornerstones of the risk assessment of new chemical entities. Standard toxicopathologic evaluation is mainly based on the study of paraffin sections stained with hematoxylin and eosin. There are, however, a number of new approaches and techniques which have improved the objectivity of evaluation and the accuracy of cell identification, and provided deeper insight into the molecular biological mechanisms of toxicity and carcinogenicity. Such approaches include the standardization of the nomenclature, the creation of data banks for morphological alterations, the use of computers to register pathological findings in toxicity studies and to statistically evaluate incidences, and the use of morphometry. Other modern techniques are immunohistochemistry, in situ hybridization, and the assessment of cell kinetics.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.